Structural investigation of a viral ortholog of human NEIL2/3 DNA glycosylases

Assault to DNA that leads to oxidative base damage is repaired by the base excision repair (BER) pathway with specialized enzymes called DNA glycosylases catalyzing the first step of this pathway. These glycosylases can be categorized into two families: the HhH superfamily, which includes endonuclease III (or Nth), and the Fpg/Nei family, which comprises formamidopyrimidine DNA glycosylase (or Fpg) and endonuclease VIII (or Nei). In humans there are three Nei-like (NEIL) glycosylases: NEIL1, 2, and 3. Here we present the first crystal structure of a viral ortholog of the human NEIL2/NEIL3 proteins, Mimivirus Nei2 (MvNei2), determined at 2.04 Å resolution. The C-terminal region of the MvNei2 enzyme comprises two conserved DNA binding motifs: the helix-two-turns-helix (H2TH) motif and a C-H-C-C type zinc-finger similar to that of human NEIL2. The N-terminal region of MvNei2 is most closely related to NEIL3. Like NEIL3, MvNei2 bears a valine at position 2 instead of the usual proline and it lacks two of the three conserved void-filling residues present in other members of the Fpg/Nei family. Mutational analysis of the only conserved void-filling residue methionine 72 to alanine yields an MvNei2 variant with impaired glycosylase activity. Mutation of the adjacent His73 causes the enzyme to be more productive thereby suggesting a plausible role for this residue in the DNA lesion search process.     

Acute in vivo treatment with 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone does not alter base excision repair activities in murine lung and liver

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a potent pulmonary carcinogen found in unburned tobacco and tobacco smoke, and is believed to play an important role in human tobacco-induced cancers. In previous studies, NNK has been reported to induce oxidative DNA damage, and to alter DNA repair processes, effects that could contribute to pulmonary tumorigenesis in rodent models. The goal of this study was to determine the effects of NNK on levels of 8-hydroxydeoxyguanosine (8-OHdG), a biomarker of DNA oxidation, and activity of base excision repair (BER), which repairs oxidative DNA damage. Female A/J mice were treated with a tumorigenic dose of NNK (10 μmol) i.p. At 1, 2 and 24 h post treatment, there were no statistically significant differences in lung or liver 8-OHdG levels between control and NNK-treated mice (P > 0.05). Furthermore, NNK did not alter lung or liver BER activity compared to control at any time point (P > 0.05). In summary, acute treatment with a tumorigenic dose of NNK did not stimulate oxidative DNA damage or significantly alter BER activity, and these effects may not be major mechanisms of action of NNK in mouse models.     

Rational design,synthesis and biological evaluation of 1,3,4-oxadiazole pyrimidine derivatives as novel pyruvate dehydrogenase complex E1 inhibitors

On the basis of previous study on 2-methylpyrimidine-4-ylamine derivatives I, further synthetic optimization was done to find potent PDHc-E1 inhibitors with antibacterial activity. Three series of novel pyrimidine derivatives 6, 11 and 14 were designed and synthesized as potential Escherichia coli PDHc-E1 inhibitors by introducing 1,3,4-oxadiazole-thioether, 2,4-disubstituted-1,3-thiazole or 1,2,4-triazol-4-amine-thioether moiety into lead structure I, respectively. Most of 6, 11 and 14 exhibited good inhibitory activity against E. coli PHDc-E1 (IC₅₀ 0.97–19.21 μM) and obvious inhibitory activity against cyanobacteria (EC₅₀ 0.83–9.86 μM). Their inhibitory activities were much higher than that of lead structure I. 11 showed more potent inhibitory activity against both E. coli PDHc-E1 (IC₅₀ < 6.62 μM) and cyanobacteria (EC₅₀ < 1.63 μM) than that of 6, 14 or lead compound I. The most effective compound 11d with good enzyme-selectivity exhibited most powerful inhibitory potency against E. coli PDHc-E1 (IC₅₀ = 0.97 μM) and cyanobacteria (EC₅₀ = 0.83 μM). The possible interactions of the important residues of PDHc-E1 with title compounds were studied by molecular docking, site-directed mutagenesis, and enzymatic assays. The results indicated that 11d had more potent inhibitory activity than that of 14d or I due to its 1,3,4-oxadiazole moiety with more binding position and stronger interaction with Lsy392 and His106 at active site of E. coli PDHc-E1.     

Microbial surface displaying formate dehydrogenase and its application in optical detection of formate

In the present work, NAD⁺-dependent formate dehydrogenase (FDH), encoded by fdh gene from Candida boidinii was successfully displayed on Escherichia coli cell surface using ice nucleation protein (INP) from Pseudomonas borealis DL7 as an anchoring protein. Localization of matlose binding protein (MBP)-INP-FDH fusion protein on the E. coli cell surface was characterized by SDS-PAGE and enzymatic activity assay. FDH activity was monitored through the oxidation of formate catalyzed by cell-surface-displayed FDH with its cofactor NAD⁺, and the production of NADH can be detected spectrometrically at 340 nm. After induction for 24 h in Luria-Bertani medium containing isopropyl-β-d-thiogalactopyranoside, over 80% of MBP-INP-FDH fusion protein present on the surface of E. coli cells. The cell-surface-displayed FDH showed optimal temperature of 50 °C and optimal pH of 9.0. Additionally, the cell-surface-displayed FDH retained its original enzymatic activity after incubation at 4 °C for one month with the half-life of 17 days at 40 °C and 38 h at 50 °C. The FDH activity could be inhibited to different extents by some transition metal ions and anions. Moreover, the E. coli cells expressing FDH showed different tolerance to solvents. The recombinant whole cell exhibited high formate specificity. Finally, the E. coli cell expressing FDH was used to assay formate with a wide linear range of 5–700 μM and a low limit of detection of 2 μM. It is anticipated that the genetically engineered cells may have a broad application in biosensors, biofuels and cofactor regeneration system.     

Novel 3-arylfuran-2(5H)-one-fluoroquinolone hybrid: Design,synthesis and evaluation as antibacterial agent

3-Arylfuran-2(5H)-one, a novel antibacterial pharmacophore targeting tyrosyl-tRNA synthetase (TyrRS), was hybridized with the clinically used fluoroquinolones to give a series of novel multi-target antimicrobial agents. Thus, twenty seven 3-arylfuran-2(5H)-one-fluoroquinolone hybrids were synthesized and evaluated for their antimicrobial activities. Some of the hybrids exhibited merits from both parents, displaying a broad spectrum of activity against resistant strains including both Gram-negative and Gram-positive bacteria. The most potent compound (11) in antibacterial assay shows MIC₅₀ of 0.11 μg/mL against Multiple drug resistant Escherichia coli, being about 51-fold more potent than ciprofloxacin. The enzyme assays reveal that 11 is a potent multi-target inhibitor with IC₅₀ of 1.15 ± 0.07 μM against DNA gyrase and 0.12 ± 0.04 μM against TyrRS, respectively. Its excellent inhibitory activities against isolated enzymes and intact cells strongly suggest that 11 deserves to further research as a novel antibiotic.     

Nucleic acid changes during photodynamic inactivation of bacteria by cationic porphyrins

Light activation of photosensitizing dyes in presence of molecular oxygen generates highly cytotoxic reactive oxygen species leading to cell inactivation. Nucleic acids are molecular targets of this photodynamic action but not considered the main cause of cell death. The in vivo effect of the photodynamic process on the intracellular nucleic acid content of Escherichia coli and Staphylococcus warneri was evaluated herein.Two cationic porphyrins (Tetra-Py⁺-Me and Tri-Py⁺-Me-PF) were used to photoinactivate E. coli (5.0 μM; 10⁸ cells mL^?1) and S. warneri (0.5 μM; 10⁸ cells mL^?1) upon white light irradiation at 4.0 mW cm^?2 for 270 min and 40 min, respectively. Total nucleic acids were extracted from photosensitized bacteria after different times of irradiation and analyzed by agarose gel electrophoresis. The double-stranded DNA was quantified by fluorimetry and the porphyrin binding to bacteria was determined by spectrofluorimetry.E. coli was completely photoinactivated with both porphyrins (5.0 μM), whereas S. warneri was only completely inactivated by Tri-Py⁺-Me-PF (0.5 μM). The hierarchy of nucleic acid changes in E. coli was in the order: 23S rRNA > 16S rRNA > genomic DNA. The nucleic acids of S. warneri were extensively reduced after 5 min with Tri-Py⁺-Me-PF but almost unchanged with Tetra-Py⁺-Me after 40 min of irradiation. The amount of Tri-Py⁺-Me-PF bound to E. coli after washing the cells is higher than Tetra-Py⁺-Me and the opposite was observed for S. warneri. The binding capacity of the photosensitizers is not directly related to the PDI efficiency or nucleic acid reduction and this reduction occurs in parallel with the decrease of surviving cells.     

Plant and fungal Fpg homologs are formamidopyrimidine DNA glycosylases but not 8-oxoguanine DNA glycosylases

Formamidopyrimidine DNA glycosylase (Fpg) and endonuclease VIII (Nei) share an overall common three-dimensional structure and primary amino acid sequence in conserved structural motifs but have different substrate specificities, with bacterial Fpg proteins recognizing formamidopyrimidines, 8-oxoguanine (8-oxoG) and its oxidation products guanidinohydantoin (Gh), and spiroiminodihydantoin (Sp) and bacterial Nei proteins recognizing primarily damaged pyrimidines. In addition to bacteria, Fpg has also been found in plants, while Nei is sparsely distributed among the prokaryotes and eukaryotes. Phylogenetic analysis of Fpg and Nei DNA glycosylases demonstrated, with 95% bootstrap support, a clade containing exclusively sequences from plants and fungi. Members of this clade exhibit sequence features closer to bacterial Fpg proteins than to any protein designated as Nei based on biochemical studies. The Candida albicans (Cal) Fpg DNA glycosylase and a previously studied Arabidopsis thaliana (Ath) Fpg DNA glycosylase were expressed, purified and characterized. In oligodeoxynucleotides, the preferred glycosylase substrates for both enzymes were Gh and Sp, the oxidation products of 8-oxoG, with the best substrate being a site of base loss. GC/MS analysis of bases released from γ-irradiated DNA show FapyAde and FapyGua to be excellent substrates as well. Studies carried out with oligodeoxynucleotide substrates demonstrate that both enzymes discriminated against A opposite the base lesion, characteristic of Fpg glycosylases. Single turnover kinetics with oligodeoxynucleotides showed that the plant and fungal glycosylases were most active on Gh and Sp, less active on oxidized pyrimidines and exhibited very little or no activity on 8-oxoG. Surprisingly, the activity of AthFpg1 on an AP site opposite a G was extremely robust with a k_obs of over 2500 min^?1.     

DNA damage induction and antiproliferative activity of vanadium(V) oxido monoperoxido complex containing two bidentate heteroligands

Several peroxidovanadium(V) complexes have been shown as a potent anticancer agents. The aim of this study was to investigate the interaction of monoperoxidovanadium(V) complex Pr₄N[VO(O₂)(ox)(phen)], (Vphen), [phen = 1,10-phenantroline, ox = oxalate(2?) and Pr₄N = tetra(n-propyl)ammonium(1+)] with DNA. UV–Vis spectrophotometry and the alkaline single-cell gel electrophoresis (SCGE, the comet assay) were used to examine the possibility of the vanadium(V) complex to induce changes in DNA. The interaction of Vphen with calf thymus DNA resulted in absorption hyperchromicity in DNA spectrum and shift of the absorption band of DNA to longer wavelengths for the [complex]/[DNA] concentration ratio equals to 4 and after 60 min of incubation. The rise in DNA absorption (by 34%) and bathochromic shift (Δλ_max = 6 nm) are indicative of the interaction between DNA and the complex molecules. DNA strand breaks in cellular DNA were investigated using the comet assay. The human lymphocytes were exposed to various concentrations of Vphen for 30 min. The results revealed that Vphen contributed to the DNA damage expressed as DNA strand breaks in concentration dependent manner. The used concentrations of Vphen (ranging from 0.1 to 100 μmol/L) caused higher DNA damage in lymphocytes compared to untreated cells (from 1.2 times for 0.1 μmol/L to 1.8 times for 100 μmol/L). Vphen was screened for its potential antitumor activity towards murine leukemia cell line L1210. Vphen exhibited significant antiproliferative activity depending on its concentration and time of exposure. The IC₅₀ values were 0.247 μg/mL (0.45 μmol/L) for 24 h, 0.671 μg/mL (1.21 μmol/L) for 48 h and 0.627 μg/mL (1.13 μmol/L) for 72 h.     

Apoptosis of tiger shrimp (Penaeus monodon) haemocytes induced by Escherichia coli lipopolysaccharide

This study was aimed at investigating the toxicity mechanism of lipopolysaccharide (LPS) on Penaeus monodon haemocytes at a cellular level. Reactive oxygen species (ROS) production, nitric oxide (NO) production, non-specific esterase activity, cytoplasmic free-Ca^2 + (CF-Ca^2 +) concentration, DNA damaged cell ratio and apoptotic cell ratio of in vitro LPS-treated haemocytes were measured by flow cytometry. Two concentrations of Escherichia coli LPS (5 and 10 μg mL^? 1) were used. Results showed that ROS production, NO production and CF-Ca^2 + concentration were significantly induced in the LPS-treated haemocytes. Ratio of DNA damaged cell and apoptotic cell increased caused by LPS, while esterase activity increased at the initial 60 min and dropped later. The initial increase in esterase activity suggested that LPS activated the release of esterase, and the later decrease might result from apoptosis. These results indicated that LPS would induce oxidative stress on shrimp haemocytes, and cause Ca^2 + release, DNA damage and subsequently cell apoptosis. This process of ROS/RNS-induced Ca^2 +-mediated apoptosis might be one of the toxicity mechanisms of LPS on shrimp haemocytes.     

Characterization of a recombinant Escherichia coli TOP10 [pQR239] whole-cell biocatalyst for stereoselective Baeyer–Villiger oxidations

This paper describes the kinetic characterization of a recombinant whole-cell biocatalyst for the stereoselective Baeyer–Villiger type oxidation of bicyclo[3.2.0]hept-2-en-6-one to its corresponding regio-isomeric lactones (−)-(1S,5R)-2-oxabicyclo[3.3.0]oct-6-en-3-one and (−)-(1R,5S)-3-oxabicyclo[3.3.0]oct-6-en-2-one. Escherichia coli TOP10 [pQR239], expressing cyclohexanone monooxygenase (CHMO) from Acinetobacter calcoaceticus (NCIMB 9871), was shown to be suitable for this biotransformation since it expressed CHMO at a high level, was simple to produce, contained no contaminating lactone hydrolase activity and allowed the intracellular recycle of NAD(P)H necessary for the biotransformation. A small-scale biotransformation reactor (20 ml) was developed to allow rapid collection of intrinsic kinetic data. In this system, the optimized whole-cell biocatalyst exhibited a significantly lower specific lactone production activity (55–60 μmol min⁻¹ g⁻¹ dry weight) than that of sonicated cells (500 μmol min⁻¹ g⁻¹ dry weight). It was shown that this shortfall was comprised of a difference in the pH optima of the two biocatalyst forms and mass transfer limitations of the reactant and/or product across the cell barrier. Both reactant and product inhibition were evident. The optimum ketone concentration was between 0.2 and 0.4 g l⁻¹ and at product concentrations above 4.5–5 g l⁻¹ the specific activity of the whole cells was zero. These results suggest that a reactant feeding strategy and in situ product removal should be considered in subsequent process design.     

Synergistic interactions in two-drug and three-drug combinations (thymol,EDTA and vancomycin) against multi drug resistant bacteria including E. coli

Combinations of two or more drugs, which affect different targets, have frequently been used as a new approach against resistant bacteria. In our work we studied the antimicrobial activity (MIC, MBC) of individual drugs (the phenolic monoterpene thymol, EDTA and vancomycin), of two-drug interactions between thymol and EDTA in comparison with three-drug interactions with vancomycin against sensitive and resistant bacteria. Thymol demonstrated moderate bactericidal activity (MBC between 60 and 4000 μg/ml) while EDTA only exhibited bacteriostatic activity over a range of 60–4000 μg/ml. MICs of vancomycin were between 0.125 and 16 μg/ml against Gram-positive and between 32 and 128 μg/ml against Gram-negative bacteria. Checkerboard dilution and time-kill curve assays were performed to evaluate the mode of interaction of several combinations against Methicillin-resistant Staphylococcus aureus (MRSA NCTC 10442) and Escherichia coli (ATCC 25922). Checkerboard data indicate indifferent interaction against Gram-positive (FICI = 1–1.3) and synergy against Gram-negative bacteria (FICI ≈ 0.4), while time kill analyses suggest synergistic effect in different combinations against both types of bacteria. It is remarkable that the combinations could enhance the sensitivity of E. coli to vancomycin 16-fold to which it is normally insensitive. We have provided proof for the concept, that combinations of known antibiotics with modern phytotherapeutics can expand the spectrum of useful therapeutics.     

Insecticidal properties of Pimpinella anisum essential oils against the Culex quinquefasciatus and the non-target organism Daphnia magna

The efficacy of an essential oil obtained from Pimpinella anisum fruits and its major compound, trans-Anethole, was tested on the eggs, larvae and adults of Culex quinquefasciatus. While causing no significant mortality on eggs, other tested stages were very sensitive to the essential oil and trans-Anethole. LC₅₀ for the 2nd to 4th instar larvae was estimated as 26–27 μL·L^− 1 and 15–19 μL·L^− 1 for the essential oil and trans-Anethole, respectively. As for the essential oil applied on adults, LC(LD)₅₀ was estimated as 9.3 μL mL^− 1 (spray test), 1.9 μL L^− 1 (fumigation test) and 0.6 μg cm^− 2 (tarsal test), and for trans-Anethole as 8.1 μL mL^− 1 (spray test), 2.1 μL L^− 1 (fumigation test) and 0.4 μg cm^− 2 (tarsal test). The time needed to achieve 50% mortality after application of LC(LD)₉₉ of the essential oil was significantly different; for example, in larvicidal assays it ranged from 15 to 235 min depending on the larval instar, and from 9 to 180 min when applied to adults, depending on the mode of application. It was also found that temperature had an important effect on the larvicidal efficacy of the essential oil, and oviposition deterrent activity was studied.The essential oil and trans-Anethole were toxic for Daphnia magna (62–92% mortality) and significantly reduced its fertility at high concentrations (35–50 μL mL^− 1) and long exposure (48 h). However, no negative effect on Daphnia mortality or fertility was found at shorter exposure times (6 h) and/or lower concentrations (20 μL mL^− 1).Based on the results of this study, we can recommend the essential oil from P. anisum as a suitable active substance for potential botanical insecticides.     

Construction of recombinant Escherichia coli for enhanced bioconversion of colchicine into 3-demethylated colchicine at 70 l bioreactor level

A recombinant strain of Escherichia coli with CYP102A1 gene was developed for the demethylation of colchicine into their derivatives. The CYP102A1 gene responsible for demethylation was isolated from Bacillus megaterium ACBT03 and amplified using suitable primers. The amplified product was cloned into pET28a+ expression vector using host E. coli BL21(DE3) cells. The CYP3A4 (product of CYP102A1 gene) protein expression and other parameters like substrate toxicity, product toxicity and enzyme activity were optimized in shake flasks; and further scaled-up to 5 l bioreactor with 3 l working volume. In 5 l bioreactor, dissolved oxygen (DO) was optimized for maximum specific growth and enhanced 3-demethylated colchicine (3-DMC) production. The optimized conditions from shake flasks were scaled-up to 70 l bioreactor and resulted into ∼80% conversion of 20 mM colchicine in 48 h with a volumetric productivity of 6.62 mg l⁻¹ h⁻¹. Scale-up factors were measured as volumetric oxygen transfer coefficient (k_La) i.e., 56 h⁻¹ and impeller tip velocity (V_tip) i.e., 7.065 m s⁻¹, respectively. The kinetic parameters K_m, k_cat, and k_cat/K_m of the CYP3A4 enzyme using colchicine as the substrate were determined to be 271 ± 30 μM, 8533 ± 25 min⁻¹, and 31.49 μM min⁻¹, respectively, when IPTG induced recombinant E. coli culture was used.     

Influence of specific growth rate over the secretory expression of recombinant potato carboxypeptidase inhibitor in fed-batch cultures of Escherichia coli

A high cell density cultivation protocol was developed for the secretory production of potato carboxypeptidase inhibitor (PCI) in Escherichia coli. The strain BW25113 (pIMAM3) was cultured in fed-batch mode employing minimal media and an exponential feed profile where the specific growth rate was fixed by limitation of the fed carbon source (glycerol). Plasmid loss rates were found to be proportional to the specific growth rate. Distribution of PCI along the cell compartments and the culture media was also dependent on the fixed growth rate. When specific growth rate was kept at μ = 0.10 h⁻¹, 1.4 g PCI L⁻¹ were obtained when adding the product present in periplasmic extracts and supernatant fractions, with a 50% of the total expressed protein recovered from the extracellular medium. This constituted a 1.2-fold increase compared to growth at μ = 0.15 h⁻¹, and 2.0-fold compared to μ = 0.25 h⁻¹. Last, a cell permeabilization treatment with Triton X-100 and glycine was employed to direct most of the product to the culture media, achieving over 81% of extracellular PCI. Overall, our results point out that production yields of secretory proteins in fed-batch cultures of E. coli can be improved by means of process variables, with applications to the production of small disulfide-bridged proteins. Overall, our results point out that control of the specific growth rate is a successful strategy to improve the production yields of secretory expression in fed-batch cultures of E. coli, with applications to the production of small disulfide-bridged proteins.     

Purification and characterization of a flavin reductase from the biodesulfurizing bacterium Mycobacterium goodii X7B

Dibenzothiophene (DBT) in fossil fuels can be efficiently biodesulfurized by a thermophilic bacterium Mycobacterium goodii X7B. Flavin reductase DszD, which catalyzes the reduction of oxidated flavin by NAD(P)H, is indispensable for the biodesulfurization process. In this work, a flavin reductase DszD in M. goodii X7B was purified to homogeneity, and then its encoding gene dszD was amplified and expressed in Escherichia coli. DszD is a homodimer with each subunit binding one FMN as cofactor. The K_m values for FMN and NADH of the purified recombinant DszD were determined to be 6.6 ± 0.3 μM and 77.9 ± 5.4 μM, respectively. The optimal temperature for DszD activity was 55 °C. DszD can use FMN or FAD as substrate to generate FMNH₂ or FADH₂ as product. DszD was coexpressed with DBT monooxygenase DszC, the enzyme catalyzing the first step of the biodesulfurization process. It was indicated that the coexpressed DszD could effectively enhance the DszC catalyzed DBT desulfurization reaction.     

Heterologous expression and characterisation of a biosynthetic thiolase from Clostridium butyricum DSM 10702

Biosynthetic thiolases (EC 2.3.1.9) are key enzymes in the branched catabolism of diverse clostridia as their activity and regulation influence the production of organic acids and solvents. In Clostridium butyricum, they are also involved in the production of hydrogen as a sustainable and environmentally benign energy source. In this study, the gene coding for thiolase from C. butyricum DSM 10702 was cloned by genome walking. It was found to consist of 1179 bp coding for a protein with 393 amino acids and a deduced molecular weight of 41.4 kDa. The enzyme was fused to an N-terminal his-tag, expressed in Escherichia coli, purified to near homogeneity and characterised for biochemical and kinetic properties. Gel filtration chromatography revealed that the catalytically active enzyme consists of a homotetramer. The enzyme showed a K_M of ～32 μM towards acetoacetyl-CoA and of ～21 μM towards CoASH at 30 °C and pH 8.0. Claisen condensation of acetyl-CoA by thiolase was analysed in a coupled enzyme assay, where β-hydroxybutyryl-CoA dehydrogenase was applied catalysing the subsequent NADH-dependant reduction of the formed condensation product acetoacetyl-CoA. For this purpose the latter enzyme was cloned from C. butyricum DSM 10702 and recombinantly expressed in E. coli. The K_M of thiolase towards acetyl-CoA was ～674 μM at 30 °C and pH 7.5. Acetyl-CoA condensation was inhibited even at micromolar concentrations of CoASH indicating that CoASH has an important regulatory function in vivo.     

Chemical composition,angiotensin I-converting enzyme (ACE) inhibitory,antioxidant and antimicrobial activities of the essential oil from south Tunisian Ajuga pseudoiva Rob. Lamiaceae

The essential oil of Ajuga pseudoiva, collected from Tunisia, was analyzed using gas chromatography–mass spectroscopy. Thirty-two compounds accounting for 95.76% of the total oil were identified. Sesquiterpenes were found to be the most abundant components of A. pseudoiva oil. And they were mainly represented by viridiflorol (30.17%), germacrene B (9.26%) (α, β and γ)-eudesmol (8.11%) and aromadendrene (7.45%). The essential oil of A. pseudoiva showed radical scavengers activity (IC₅₀ = 0.72 mg/mL) and displayed lipid peroxidation inhibitory activity (IC₅₀ = 0.6 mg/mL). A. pseudoiva essential oil was also found to exhibit a dose-dependent ACE inhibitory activity with an IC₅₀ value of 65.5 μg/mL. Moreover, the antimicrobial activity of the essential oil was tested against 17 species of microorganisms, and the results obtained showed significant antibacterial activity against the Gram-positive and Gram-negative bacteria, with inhibition zones and minimal inhibitory concentration values of 14–32 mm and 84–137 μg/mL and 6–21 mm and 105–336 μg/mL, respectively. Higher activity was also found against several fungal strains.     

Discovery,characterization and comparison of inhibitors of Bacillus anthracis and Staphylococcus aureus replicative DNA helicases

Antibacterial compounds with new mechanisms of action are needed for effective therapy against drug-resistant pathogens in the clinic and in biodefense. Screens for inhibitors of the essential replicative helicases of Bacillus anthracis and Staphylococcus aureus yielded 18 confirmed hits (IC₅₀ ? 25 μM). Several (5 of 18) of the inhibitors were also shown to inhibit DNA replication in permeabilized polA-deficient B. anthracis cells. One of the most potent inhibitors also displayed antibacterial activity (MIC ～5 μg/ml against a range of Gram-positive species including bacilli and staphylococci) together with good selectivity for bacterial versus mammalian cells (CC₅₀/MIC > 16) suitable for further optimization. This compound shares the bicyclic ring of the clinically proven aminocoumarin scaffold, but is not a gyrase inhibitor. It exhibits a mixed mode of helicase inhibition including a component of competitive inhibition with the DNA substrate (K_i = 8 μM) and is rapidly bactericidal at 4 × MIC.     

Design,synthesis, antiviral and cytostatic activity of ω-(1H-1,2,3-triazol-1-yl)(polyhydroxy)alkylphosphonates as acyclic nucleotide analogues

The efficient synthesis of a new series of polyhydroxylated dibenzyl ω-(1H-1,2,3-triazol-1-yl)alkylphosphonates as acyclic nucleotide analogues is described starting from dibenzyl ω-azido(polyhydroxy)alkylphosphonates and selected alkynes under microwave irradiation. Selected O,O-dibenzylphosphonate acyclonucleotides were transformed into the respective phosphonic acids. All compounds were evaluated in vitro for activity against a broad variety of DNA and RNA viruses and for cytostatic activity against murine leukemia L1210, human T-lymphocyte CEM and human cervix carcinoma HeLa cells. Compound (1S,2S)-16b exhibited antiviral activity against Influenza A H3N2 subtype (EC₅₀ = 20 μM—visual CPE score; EC₅₀ = 18 μM—MTS method; MCC >100 μM, CC₅₀ >100 μM) in Madin Darby canine kidney cell cultures (MDCK), and (1S,2S)-16k was active against vesicular stomatitis virus and respiratory syncytial virus in HeLa cells (EC₅₀ = 9 and 12 μM, respectively). Moreover, compound (1R,2S)-16l showed activity against both herpes simplex viruses (HSV-1, HSV-2) in HEL cell cultures (EC₅₀ = 2.9 and 4 μM, respectively) and feline herpes virus in CRFK cells (EC₅₀ = 4 μM) but at the same time it exhibited cytotoxicity toward uninfected cell (MCC ⩾ 4 μM). Several other compounds have been found to inhibit proliferation of L1210, CEM as well as HeLa cells with IC₅₀ in the 4–50 μM range. Among them compounds (1S,2S)- and (1R,2S)-16l were the most active (IC₅₀ in the 4–7 μM range).     

Synthesis and antimicrobial activity of polyhalo isophthalonitrile derivatives

A series of polyhalo isophthalonitrile derivatives (3 and 4) that incorporate a variety of substituents at the 2-, 4-, 5- and/or 6-positions of the isophthalonitrile moieties have been designed and synthesized. These derivatives were evaluated for their antimicrobial activity against Staphylococcus aureus, Bacillus cereus (Gram-positive bacteria), Escherichia coli, Pseudomonas aeruginosa (Gram-negative bacteria); and Candida albicans (Fungi). Compounds 3 and 4 showed stronger inhibition of Gram-positive bacteria and fungi growth, and the antimicrobial ability of compound 3j (a 4-(benzylamino)-5-chloro-2,6-difluoro analog, MIC[SA] = 0.5 μg/mL; MIC[BC] = 0.4 μg/mL; MIC[CA] = 0.5 μg/mL) were close to nofloxacin and fluconazole and identified as the most potent antimicrobial agents in the series. The preliminary analysis of structure–activity relationships is also discussed.